DNA Repair Flashcards
relationship btwn mutations, DNA repair and cancer
Changes in DNA molecules can cause mutations. After replication, these changes result in a permanent alteration of the base sequences in the daughter DNA. Cancer and many human diseases are the consequence of mutation due to DNA damage and inadequate DNA repair.
examples of heritable human diseases caused by defective DNA repair pathways
- Mutations in genes that mediate NER lead to human genetic diseases including Cockayne Syndrome (CS), Xeroderma pigmentosum (XP), and Trichothiodystrophy (TTD).
- Hereditary non-polyposis colorectal cancer (HNPCC) is caused by mutations in the machinery that performs mismatch repair in humans.
DNA damage: change of base
- Spontaneous base loss (depurination, depyrimidination)
- Spontaneous deamination (A, C, G)
- Sunlight (UV)-induced thymidine dimers
- Base alkylation (e.g. O6-meG)
- Base oxidation by reactive oxygen species (e.g. 8-oxo-dG)
DNA damage: change in DNA structure
Bulges in DNA double helix caused by:
Insertion/deletion of nucleotides Bulky chemical adducts Replication errors (mismatch) Intra/inter-strand crosslinks
• DNA strand breaks
• Stalled DNA replication forks
Damage that can occur can cause change-causing mutations
oxidative damage caused
by products of normal metabolic activity of the cell, cleavage of a DNA strand caused by
radiation and chemicals, chemical alterations to the base (e.g. alkylation), loss of a base
(depurination / depyrimidination), loss of an amine group of the base (deamination), sunlight
induced thymine-dimers
Machinery used to repair damage
- direct
- excision
- base excision repair
- nucleotide excision repair
- mismatch repair
- lesion bypass
Direct repair
(ex. ligation of a break in the phosphodiester backbone of the DNA by DNA ligase or repair by MGMT)
-Excision Repair:
excision of the damaged region, followed by precise replacement. Basically all excision repair pathways require endonuclease and/or exonuclease, DNA polymerase and DNA ligase.
—Base excision repair (BER):
Repairs base damage that does not distort the DNA and is initiated by damage-specific glycosylases that release the damaged base, followed by removal of the damaged nucleotide.
—Nucleotide excision repair (NER):
removes DNA lesions that distort the DNA structure and block RNA or DNA polymerase movement on the DNA (ex. thymidine dimers, carcinogen induced bulky additions to bases). A short oligonucleotides (13-30 nt) including the damaged base are removed via dual incision by two endonucleases. Has two modes of damage recognition
- Transcription Coupled NER, or
- Global Genome NER. Mutations in NER machinery causes CS or/ and XP conditions
—Mismatch repair (MMR):
Removes nucleotides that are misincorporated during DNA replication.New strand of DNA is recognized by hemimethylation state in E. coli, and by nicks on newly synthesized DNA strand in humans.Mutations in the MMR machinery cause HNPCC
-Lesion bypass:
Is used when cells have too much DNA damage for the “error-proof” repair machineries (NER, BER, MMR) to handle, especially replication-blocking lesions, and cells employ “bypass” or “error-prone” DNA polymerases with loosened specificity to enable replication to continue through damaged template strand (The bypass polymerases lack the proofreading 3’-to-5’ exonuclease activity and have error rates 100-10,000 fold higher than those of the polymerases used in normal DNA replication)
Double stranded DNA breaks (DSBs) can be repaired by _______ or _______
by either non-homologous end
joining (NHEJ) or homologous recombination (HR)
Molecular consequences of DNA damage
- thymine dimers (from UV damage)
- uracil mis-incorportations
- bulky chemical adducts
- double strand breaks
Steps common to all 3 excision repair mechanism
- recognition of damaged/mismatched nucleotide
- Endonuclease-mediated cutting of phosphdiester backbone flanking the damage
- Nuclease mediated removal of DNA fragment containing damaged/mismatched nucleotide
- DNA poly mediated synthesis of missing dNTPS
- DNA ligase sealing the remaining nick of phosphodiester backbone
Steps of NER
- Recognition and binding of the damaged site by a multi-protein complex (two different ways depending on local transcription activity).
- Local unwinding of the DNA duplex by helicases (parts of the TFIIH protein complex) to form a bubble of ~25 bases.
- Double incision of the damaged strand By two endonucleases and removal of a ~30 base oligonucleotide containing the lesion.
- Filling in the gap by a DNA polymerase. 5. Rejoining the two ends by a DNA ligase.
Nucleotide excision repair (NER) removes damage that
distorts the DNA structure and blocks poly function
BER is initiated by
glycosylase, which flips out the damaged base from the stacked region of DNA duplex and removes it by hydrolyzing the glycosidic bond
Base excision Repair removes base damage that
doesn’t distort DNA duplex
BER steps
Step 1: Modified base is recognized by a specific DNA glycosylase, which hydrolyzes the N-glycosidic bond, yielding an AP site.
Step 2. An AP site-specific endonuclease (APE1) cleaves the sugar-phosphate backbone 5’ to the AP site.
Step 3. Another endonuclease cuts 3’ to the AP site, removing the deoxyribose phosphate.
Step 4. The resulting gap is filled by DNA polymerase, and the nick sealed by DNA ligase.
Two ways NER recognize damage
Global genome NER
Txn coupled NER
Global genome NER
recognize damage anywhere in genome
defects cause cancer, like XP
Txn-coupled NER
recognizes damage within transcribed region
defects cause CNS disorder
cockayne sundrom (CS)
MMR corrects errors made by
DNA poly during replication
MMR is initiated by
two protein complexes
MutS homolog: hMSH
MutL Homologs: hMLH and PMS
how does MMR know which strand is old vs new
Nascent lagging strand is marked by transient 5’ DNA ends of short discontinuous Okazaki fragments.
Nascent leading strand is marked by transient presence of ribonucleotides (1 rNMP/1,250 dNMP), which is processed into nicks by RNase H2.
PARP: Poly(ADP-ribose) polymerase is activated by a
by a single-strand break and adds poly(ADP-ribose) chains to proteins.
________ of proteins near a single strand break site facilitates DNA repair
Reversible Poly (ADP-ribosylation)
PARP’s role in SSB repair:
- amplification of damage signal
- Focal enrichment of repair proteins
- change in local chromatin structure
DNA damage checkpoint
signals–> sensors–> transducers (kinases) –> effectors
-Lesion bypass (aka translesion synthesis):
If a cell encounters so much DNA damage of the type that normally blocks DNA replication (such as UV-induced thymidine dimers) that the excision repair systems cannot fix it all, cells
resort to this pathway. As a last resort, cells employ “bypass” or “error-prone” DNA polymerases with loosened specificity to enable replication to continue through damaged template strand. However, it is highly mutagenic because alternate DNA polymerases that lack 3’ to 5’ proofreading exonuclease activity are used to replicate past the DNA lesion, resulting in an error rate 100-10,000 higher than normal DNA replication.
DNA Damage Checkpoint halts
DNA Damage Checkpoint halts cell cycle progression when DNA is compromised to allow time for DNA repair. It is a signaling pathway composed of damage sensors, signal transducers and effectors. Protein kinases ATM and ATR are recruited to initiate the sequential recruitment and activation of downstream proteins (and delay or prevent cancer in earlier stages of tumorigenesis).
Changes in DNA molecules can cause ___________
mutations
__________ are the consequence of mutation due to DNA damage and inadequate DNA repair.
cancer and many human diseases
Mutations in genes that mediate NER lead to human genetic diseases including ________ , ________, and ________.
Cockayne Syndrome (CS) Xeroderma pigmentosum (XP) Trichothiodystrophy (TTD).
